A GATA-1-Regulated microRNA Cluster That Is Essential for Erythropoiesis.

The nuclear factor GATA-1 controls erythropoiesis by regulating transcription of numerous protein-encoding genes. Here we show that GATA-1 also activates the expression of a critical erythroid microRNA (miR) locus. MicroRNAs regulate gene expression and tissue differentiation by binding the 3′ untranslated regions of mRNA transcripts to inhibit their translation or stability. We used an oligonucleotide-based microarray to analyze miR expression in G1E cells, a GATA-1-null erythroid line that undergoes terminal maturation when GATA-1 activity is restored. We identified 8 miRs that are significantly upregulated during GATA-1-induced erythroid maturation. Two of the most highly induced miRs, 144 and 451, are encoded in tandem on mouse chromosome 11 and are transcribed on the same primary microRNA transcript. Northern blot studies showed that the miR144/451 locus is strongly induced by GATA-1 and specifically expressed at high levels in human and murine erythroid precursors. Bioinformatic analysis of the miR144/451 locus identified a conserved region containing three closely-spaced GATA binding motifs located about 2.7 kb upstream of the transcriptional start. In erythroid cells, this region exhibits enhancer activity and binds GATA-1 and its cofactor FOG-1, as determined by chromatin immunoprecipitation studies. MicroRNAs 144 and 451 are conserved between mammals and zebrafish, facilitating further in vivo studies. Whole mount in situ hybridization of zebrafish embryos showed both miRs to be exclusively expressed in the developing blood islands (intermediate cell mass) at 24h post-fertilization (hpf), colocalizing with gata-1 and β-globinE1 expression. Both miRs are deficient in the gata-1 null mutant, vlad tepes, consistent with their epistatic relationship to gata-1. Transient inactivation of miR-451 function using anti-sense morpholino oligomers selectively ablated hemoglobin staining cells in embryos at 48 hpf. These morphant embryos also exhibited dramatically reduced β-globinE1 mRNA expression. In contrast, there were no quantitative effects on thrombocytes (platelet-equivalent cells) in morphant embryos. Together, our data define a new regulatory axis through which GATA-1 promotes red cell development by directly activating the transcription of miR144/451, a critical erythroid-specific microRNA locus.